EZ1 DNA Investigator Kit

For automated purification of DNA from 1–6 or 1–14 forensic and human identity samples per run

More efficient yields from trace casework samples

Higher signal-to-noise ratios

“Tip dance” protocol for processing solid material

Optional TE elution and use of carrier RNA

The EZ1 DNA Investigator Kit reproducibly automates purification of genomic DNA from reference and casework samples in human identity testing. The kit can be used with the EZ1 Advanced XL (1–14 samples per run), EZ1 Advanced (1–6 samples per run), and BioRobot EZ1 (1–6 samples per run) instruments. Purification is fast and efficient, and purified DNA performs well in downstream analyses, such as quantitative PCR and STR analysis, with high signal-to-noise ratios.

Papers from cigarette butts or 3 paper disks per sample were spotted with 50 ng DNA per sample. After proteinase K digestion, the samples were incubated at 95°C for 5 minutes. Solid materials were removed from half of the samples, which were then processed using the EZ1 DNA Investigator Kit with the standard trace protocol (Trace). The remaining half of the samples was processed using the EZ1 DNA Investigator Kit with the "tip dance" protocol, without removing solid materials from the sample tubes (Tip dance). DNA yields were quantified by real-time, quantitative PCR.|DNA was diluted in Buffer G2 to a final concentration of 50 pg/μl, and the indicated volumes were processed using the EZ1 DNA Investigator Kit with the standard trace protocol (Trace) or the large-volume protocol (LV). All samples were eluted in 50 μl water, and 5 μl was quantified using real-time, quantitative PCR.|In addition to the standard trace protocol, an optional, fully automated "tip dance" protocol can be used, where the filter-tip moves back and forth relative to the worktable platform while pipetting. This enables processing of solid materials, such as swabs, fabrics, blood discs, or cigarette butts, directly in the sample tube. There is no need for prior centrifugation to remove solid materials that could clog the tip.|AmpFlSTR control DNA (1 ng) was diluted in 200 μl Buffer G2 and purified using the EZ1 DNA Investigator Kit and the trace protocol on the EZ1 Advanced workstation. DNA was eluted in 50 μl water, and 10 μl (corresponding to 200 pg DNA) was used for STR analysis. PCR products were analyzed on an ABI PRISM 310 Genetic Analyzer with Genotyper software. (Data kindly provided by B. Bayer and K. Anslinger, Institute of Legal Medicine, Ludwig Maximilian University, Munich, Germany.)|

More efficient yields for sensitive downstream analyses

The EZ1 DNA Investigator Kit provides optimized chemistries for automated DNA purification with more efficient yields. STR analysis shows higher peaks for more sensitive detection (see figure "Improved performance in STR analysis", see table "Relative STR peak heights with different protocols"). The higher peaks demonstrate the improved signal-to-noise ratio for highly sensitive detection and high performance in downstream applications. Comparison of peak heights in STR analysis showed recovery of 79% of the potential DNA yield (not shown). Optional elution in TE buffer gives comparable performance (see table "Relative STR peak heights with different protocols") and provides increased stability for long-term storage of small amounts of purified DNA. There were no dropouts (<50 relative fluorescent units for any of the 8 alleles investigated) for any of the samples processed (see table "Relative STR peak heights with different protocols").

Relative STR peak heights with different protocols

Experiment

Kit and card

Protocol

Elution

Relative peak height

Number of dropouts

1

EZ1 DNA Tissue Kit and EZ1 DNA Forensic Card

Trace

Water

100%

0/8

1

EZ1 DNA Investigator Kit and Card

Trace

Water

176%

0/8

2

EZ1 DNA Investigator Kit and Card

Trace

Water

100%

0/8

2

EZ1 DNA Investigator Kit and Card

Trace

TE

116%

0/8

3

EZ1 DNA Investigator Kit and Card (200 µl)

Trace

Water

100%

0/8

3

EZ1 DNA Investigator Kit and Card (500 µl)

Large-volume

Water

203% (81% normalized to starting volume)

0/8

AmpFlSTR control DNA was diluted in Buffer G2 to a final concentration of 5 pg/µl, and 200 µl (or 500 µl for the large-volume protocol) was processed using the indicated kits and protocols, with 50 µl elution volumes. Peak heights for 8 peaks were normalized within each experiment. (Data kindly provided by B. Bayer and K. Anslinger, Institute of Legal Medicine, Ludwig Maximilian University, Munich, Germany.)

Easier handling of solid materials with "tip dance" protocol

In addition to the standard trace protocol, an optional, fully automated "tip dance" protocol can be used, where the filter-tip moves back and forth relative to the worktable platform while pipetting. This enables processing of solid materials, such as swabs, fabrics, blood discs, or cigarette butts, directly in the sample tube. There is no need for prior centrifugation to remove solid materials that could clog the tip (see flowchart "EZ1 DNA tissue trace and “tip dance” protocols"). The "tip dance" protocol simplifies handling and, in some cases, increases DNA yield with more efficient extraction of the sample (see figure "Easy and efficient processing of solid materials with the "tip dance" protocol").

Optimized protocol for larger sample volumes

A large-volume protocol enables fully automated processing of starting volumes up to 500 µl. This not only allows efficient DNA purification from dilute samples with low concentrations of DNA, such as diffuse stains, but also enables purification from samples that require larger volumes for thorough lysis. The ability to process larger sample volumes — with the same elution volume as the standard trace protocol — enables higher yields of more concentrated DNA for greater sensitivity in downstream applications (see figure "Higher yields of concentrated DNA with the large-volume protocol", see table "Relative STR peak heights with different protocols").

Efficient purification of low amounts of DNA

The EZ1 DNA Investigator Kit now includes carrier RNA for increased efficiency. Carrier RNA enhances binding of DNA to the silica surface of the magnetic particles, especially if the sample contains low amounts of DNA (<100 ng). Published data suggest that addition of carrier RNA enables more efficient isolation of low amounts of DNA from forensic samples and may, for some sample types, provide improved DNA yields. Addition of carrier RNA to sample lysates did not interfere with downstream STR analysis.

Easy protocol setup and handling

The preprogrammed EZ1 Advanced XL DNA Investigator Card (for use with the EZ1 Advanced XL), EZ1 Advanced DNA Investigator Card (for use with the EZ1 Advanced), and EZ1 DNA Investigator Card (for use with the BioRobot EZ1) make setting up the purification protocols as easy as making a credit card transaction. Prefilled, sealed reagent cartridges in the EZ1 DNA Investigator Kit help to prevent human error during setup of the instrument and prevent contamination of buffers. By eliminating human error in reagent preparation and workstation setup, and by providing cross-contamination-free liquid handling, the EZ1 Advanced XL, EZ1 Advanced, and BioRobot EZ1 provide high levels of reliability and standardization.

Pretreated samples are loaded onto the instrument, and genomic DNA is purified in one step following the automated EZ1 DNA procedure. Genomic DNA is eluted, ready to use in all downstream applications.

Applications

The high-quality DNA obtained using the EZ1 DNA Investigator Kit is suited for human identity applications including forensics, kinship or paternity testing, and disaster victim identification, and is used in techniques such as:

This protocol has been adapted by the North Louisiana Criminalistics Laboratory from the pretreatment for bone or teeth and is for the lysis and extraction of DNA from forensic samples of calcified tissue in less time than the standard EZ1 DNA Investigator protocol pretreatment.

This protocol has been adapted by the North Louisiana Criminalistics Laboratory from the pretreatment for hair and is for the lysis and extraction of DNA from forensic samples of hair in less time than the standard EZ1 DNA Investigator protocol pretreatment.

This protocol has been adapted by the North Louisiana Criminalistics Laboratory from the pretreatment for epithelial cells mixed with sperm cells and is for the lysis and extraction of DNA from a sperm pellet after differential separation.

This protocol has been adapted by the North Louisiana Criminalistics Laboratory from the pretreatment for bone or teeth and is for the lysis and extraction of DNA from forensic samples of calcified tissue in less time than the standard EZ1 DNA Investigator protocol pretreatment.

This protocol has been adapted by the North Louisiana Criminalistics Laboratory from the pretreatment for hair and is for the lysis and extraction of DNA from forensic samples of hair in less time than the standard EZ1 DNA Investigator protocol pretreatment.

This protocol has been adapted by the North Louisiana Criminalistics Laboratory from the pretreatment for epithelial cells mixed with sperm cells and is for the lysis and extraction of DNA from a sperm pellet after differential separation.

This protocol has been adapted by the San Diego Police Department (SDPD) Crime Laboratory from the standard QIAGEN pretreatment for casework samples, and is intended for the lysis and extraction of DNA from forensic samples of fired (casings) and unfired (cartridges) ammunition.